Many enzymes containing redox-active metal centers play significant roles in cellular function, and are often involved in a variety of physiologically important processes. In particular, several Mn-containing metalloproteins have emerged with functional roles in O2 metabolism since the identification of Mn as an essential metal in biological redox catalysis. These include a mitochondrial Mn-superoxide dismutase (SOD2) that detoxifies superoxide radicals into O2 and peroxide; a non-heme Mn-containing pseudocatalase that catalyzes the decomposition of peroxide into H2O and O2; and the oxygen-evolving complex (OEC) in photosystem II (PSII), which is possibly the most important due to its key role in the oxidation of H2O to O2 during photosynthesis. Nearly all of the atmospheric O2 that supports aerobic life is produced and replenished by the OEC through H2O oxidation; hence, this light-induced reaction is one of the most important biological redox processes found in nature. Although it is known that the OEC is composed of a heteronuclear Mn4CaOx cluster where four electrons are extracted in a stepwise manner from two H2O molecules to produce one O2 molecule, the detailed structure and mechanism of how this process occurs are not well understood. Furthermore, conventional X-ray crystallography and spectroscopy approaches are limited by the sensitivity of the redox-active metal complex to radiation damage by photoreduction. However, the recent development of the powerfully intense X-ray free electron laser (X-FEL) and application of the collect before destroy approach provide a viable option for overcoming this obstacle. Thus, a key objective of this proposal is to determine the structure of the intact OEC and elucidate the catalytic mechanism by which H2O is oxidized to O2 by mapping the time evolution of the Mn4CaOx cluster using this new X-FEL technology. Specifically, X-ray diffraction (XRD) and X-ray emission spectra (XES) will be simultaneously measured from a continuous stream of PSII microcrystals with femtosecond X-FEL pulses in order to determine not only the electronic and geometric structure of the Mn4CaOx cluster, but also the integrity of the metal complex. Two fundamental points that are central to understanding photosynthetic water oxidation include: (i) the temporal evolution of the OEC electronic structure, and (ii) the structural dynamics in the ligand environment and Mn4CaOx cluster as it cycles through the catalytic steps. To address these points and map the light-induced chemical steps in real time, a combined laser excitation 'pump' and X-FEL 'probe' with variable time delays will be incorporated into the experimental setup. Not only will this study lead to an understanding of the mechanism of H2O oxidation to form O2, but the methodology developed here should also have broad applications as a model study for using X-FELs to determine structure and dynamics in other physiologically important membrane proteins and redox- active metalloenzymes that are prone to X-ray radiation damage.